The selective recognition, sorting, and transport of proteins from one intracellular organelle to another via vesicular carriers represents an essential feature of all eukaryotic cells. The precise mechanisms and machinery that direct these processes are not yet known. We have isolated a large collection of mutant that exhibit severe defects in protein delivery to the yeast lysosome-like vacuole. The defective genes in these mutant are likely to encode components of the cells' protein sorting apparatus. Toward an understanding of the molecular and biochemical basis of the vacuolar protein sorting defects in these mutants (vps), we propose to characterize the roles certain of the VPS genes and their products play in the sorting and delivery of vacuolar enzymes from the Golgi complex to the vacuole in yeast. The fundamental similarities between yeast and other eukaryotic cells in their pathways for protein delivery, together with the powerful genetic and molecular approaches available in yeast, make yeast an ideal organism for addressing these problems. The yeast mutants we have isolated exhibit a similar phenotype to many transformed mammalian cell lines that are known to aberrantly secret lysosomal proteases. The mislocalization of these proteases has been proposed to potentiate the metastasis of tumor cells. The proposed studies focus on two of the VPS genes, VPS15 and VPS34. Mutations in each gene result in a common set of phenotypes suggesting that each may facilitate the same step in the vacuolar protein delivery pathway. The sequence of the VPS15 gene predicts a protein with the following features: a concensus site for N-terminal myristoylation, a region of homology with the regulatory subunit of protein phosphatase 2A (PP2A). The VPS34 gene product shares significant sequence similarity with the catalytic subunit of mammalian phosphatidylinositol-3-kinase. VPS34 P13-kinase activity may be regulated by the VPS15 gene product. We have found that overexpression of the Vps34 protein can suppress the defects caused by mutation within the kinase domain of the VPS15 gene product, but will not suppress a null allele of VPS15. Using both genetic and biochemical approaches, we propose to characterize: 1) the functional role of the Vps15 kinase domain, as well as protein phosphorylation, in vacuolar protein sorting, 2) the significance of the sequence homology in Vps15p to the PP2A regulatory subunit, 3) the subcellular location and possible site of action for both the Vps15 and Vps34 proteins, and 4) the potential interaction of Vps15 and Vps34 proteins with each other and possibly other cellular components (e.g., PP2A).